How To Perform HPLC Peak Purity Using PDA Detector
Peak purity by PDA detector is a widely used technique to check the HPLC peak purity in the pharmaceutical industry due to its simplicity and fast result. In this article, you will learn about the peak purity concept, different methods for calculating peak purity, advantages, limitations, and applications of peak purity. You will also learn the concept of the PDA detector. After reading the article your doubts will be cleared and you will be able to answer several questions related to peak purity and PDA detector.
Table of content
- Introduction and Outcome
- PDA or DAD Detector
- Peak Purity concept
- Difference between PDA and UV detector
- Difference between Peak purity and Chemical purity
- 3 Widely used peak purity calculation techniques
- Peak purity terminology
- HPLC Peak Purity by PDA Detector: How Authentic?
- Limitations of Peak Purity
- Advantages/Applications of Peak Purity Test
- Conclusion
- FAQs
What is the peak purity?
The process of assessing the spectral uniformity of a chromatographic peak is called the peak purity.
Why is peak purity needed?
HPLC is used at almost all stages during pharmaceutical development to control the quality of APIs. It may be possible that other impurity peaks may co-elute with the peak of interest. Peak purity is performed to check the covariation/interference of other peaks with the peak of interest. In the following HPLC chromatogram (Figure1) impurity A and impurity B are coeluting with the main peak
Figure
PDA or DAD Detector
PDA detector or photodiode array detector acquires absorbance data over the entire uv-visible range (190 nm to 900 nm). Therefore an analytical scientist can extract chromatograms at multiple wavelengths within the selected wavelength range as per requirement during method development. Peak spectra can also be generated as per the required wavelength range. It is very helpful during HPLC method development. It is also called DAD or Diode array detector.
PDA wavelength range
Most PDA instruments are available with wide UV-visible ranges such as 190 to 900nm. But some of the instruments may have narrow UV ranges like 190 nm to 650 nm or 200 nm to 650 nm
Advantages and Applications of PDA Detectors
The following are the advantages of the PDA detector:
- To know peak maxima in HPLC chromatogram
- To know the peak homogeneity or peak purity
- It tells about the coelution of the peak
- To decide operational wavelength during HPLC method development
- It is cost effective
- It gives fast result
- Very helpful during HPLC method development
Limitations of PDA detector
The following are the limitations of the PDA detector:
- Not suitable for molecules having no UV absorption
- Less suitable for related substances or impurity profile testing due to less sensitivity
- Not suitable for inorganic compounds
- A PDA detector is low sensitive detector
Peak Purity concept
The process of assessing the spectral uniformity of a chromatographic peak is called the peak purity. This is the analysis of absorption spectra across peaks to determine whether they are all similar or have differences. It tells about the coelution of the peak in the main peak. This concept is based on the UV principle.
Peak Purity by PDA Detector
Peak purity by PDA detector is based on the comparison of spectra recorded during the elution of the peak (see Figure-1). Typically, the following three spectra are taken and compared to assess the peak purity by the PDA detector:
- Two spectra at the inflexion points (upslope and downslope spectrum) and
- One at the apex (see Figure 2).
All the above three spectra satisfy Peak purity suitability criteria.
Figure-2
Note: If required one can select more than three points for spectra generation and can use all spectra for peak purity calculation.
Peak purity suitability criteria
- If all the above three spectra are similar or match each other then the peak theoretically has no spectral impurities and the peak is said to be pure.
- If the above three spectra are not similar or do not match each other then theoretically the peak contains spectral impurity and then the peak is said to be impure
In the figure-3:
- All the three-point spectra (upward slope, peak apex and downward slope) of peak A are matching and hence peak A is pure
- All the three-point spectra (upward slope, peak apex and downward slope) of peak B are not matching and hence peak B is impure
Factors affecting peak purity
Peak purity test may be affected:
- If close eluting peaks are not separated base to base
- Noise or background absorption
- If spectral absorption co-eluting peaks are low of
- If the impurity and main compound have the same or almost the same spectra with similar elution time
Sample Concentration optimization for Peak Purity
- Sample concentration should be adjusted in such a way that absorbance should be below 1000 mAu
- Peak must-have area response value ≥ QL
Peak Purity Test Wavelength-Range Setting
Wavelength selection plays an important role in peak purity determination. If wavelength has not been chosen scientifically then the peak purity result may be incorrect. The following factors must be considered while selecting the wavelength range:
- Buffer concentration: Higher buffer concentration in the mobile phase may affect peak purity. Always put a lower wavelength above the buffer UV cut-off, If possible.
- Operating wavelength: If the operating wavelength is 254 nm and all impurities and the main analyte have maxima ≥ 240 nm then select a wavelength range like 220 nm to 350 nm and not like 190 nm to 650 nm
- Buffer quality: Poor buffer quality may lead to failure of peak purity due to formation of noise. Always use HPLC grade buffer
- Type of buffer: Peak purity also depends upon the type of buffer. If buffer-like ion pairs (such as Hexane sulfonic acid sodium salts, tetrabutylammonium hydroxide etc) are used in that case keep the lower wavelength range limit more than the UV cut-off of the buffer
- Solvent quality: Poor quality of solvent may lead to failure of peak purity. Always use gradient grade HPLC solvent while determining peak purity
Difference between PDA and UV detector
| PDA | UV detector |
| In a PDA detector, wavelength range is given and hence chromatogram can be generated at any wavelength as per the requirement | In a UV detector, the operating wavelength (one or two as per instrument provision) is given and hence chromatogram can be generated only at one or two wavelength |
| It can be used to determine both peak purity and chemical purity | It can only be used to determine chemical purity |
Difference between Peak purity and Chemical purity
| Peak purity | Chemical purity |
| Peak purity tells about the homogeneity or purity of the chromatographic peak | Chemical purity tells about the chemical purity of the main molecule |
| It depends on those impurities which elute with the main peak. The impurities which are separated from the main peak can not affect the peak purity. | It depends on the impurities present in the molecules or each peak present in the chromatogram |
| It depends on noise, solvent composition, buffer concentration and co-elution of two of more than two peaks | It does not depend on noise, solvent composition and buffer concentration |
| It tells about the authenticity of the HPLC method | It tells about the quality/authenticity of the molecule |
| It is determined by PDA or mass detector | It is determined by HPLC, GC, Chemical titration, FTIR, NMR etc |
3 Widely used peak purity calculation techniques
The following techniques are widely used for peak purity:
- Spectral normalisation method
- Spectral similarity method and
- Ratio gram generation method
Among the above methods, the spectral normalisation method and Spectral similarity method are widely used in industries for peak purity calculation
Spectral normalisation method
This technique involves normalising and comparing spectra taken across different points of the peak. This technique involves following steps;
- First spectra are acquired at the upslope, the apex and the downslope and
- The spectra are normalised and overlaid for graphical presentation
Peak purity suitability criteria
- If all the above three spectra are similar or match each other then the peak theoretically has no spectral impurities and the peak is said to be pure.
- If the above three spectra are not similar or do not match each other then theoretically the peak contains spectral impurity and the peak is not said to be pure
This technique very good for interactive data evaluation and for automated routine analysis
Figure-3
- In the above HPLC chromatogram; The spectra of all the points on the peak match across all points for peak A and hence peak A is spectrally pure.
- The spectra of all the points on the peak are matching across all points for peak B and hence peak B is spectrally impure.
Spectral Similarity
In this technique, all spectra from the concerned peak are compared with one or more selected spectra (reference spectra). Generally, the apex spectrum is taken as the reference spectrum. The degree of match or spectral similarity is plotted over time during elution.
The ideal profile for a pure peak is a flat line and it is denoted as 1000. It is called similarity curve.
At the start point and end point of each peak, spectra may vary due to the contribution of background noise take place. Due to this noise interference flat line is not always possible. That is the reason the threshold is kept. Generally, 980 is set as the threshold value for peak purity calculation.
Spectral Similarity peak purity acceptance criteria
The following criteria must be satisfied for peak purity:
- If the peak purity factor or purity angle is within the threshold value or purity threshold, the peak is classified as pure
- If the peak purity factor exceeds the threshold value, the peak is classified as impure.
Peak purity terminology
Match factor
The spectral comparison of the two spectra is called the match factor. The following are the relationships between match factor and spectra similarity:
- Match factor = 0, indicates no matching between the two spectra
- Match factor = 1000, indicates 100% matching between two spectra (identical spectra)
- Match factor = 990, which indicates two spectra are similar
- Match factor = 900 to 990, which indicates some similarity between the two spectra
- Match factor = < 900, which indicates two spectra are different
Purity factor or Purity level
The purity factor is the mean value of the match factor. It is the measure of the similarity in the shape of the spectra of a given peak.
HPLC Peak Purity by PDA Detector: How Authentic?
The peak purity by PDA detector is not a foolproof technique for peak purity checking of any peak. It has the following limitations and advantages:
Limitations of Peak Purity
The following are the limitations of the peak purity test:
- The coeluting impurity must have UV absorbance
- The coeluting impurity must have a value more than the quantification level (QL)
- The coeluting impurity must have spectra different from the main analyte
- The coeluting impurity must not be the inorganic compounds
- The coeluting impurity must not be the optical isomer
Advantages/Applications of Peak Purity Test
The following are the advantages/applications of the peak purity test:
- Covers both UV and visible range (200 nm to 900 nm)
- Very helpful during method development and forced degradation studies
- Cost effective technique
- Fast analysis
- Tells about stability indicating nature of the HPLC method
PDA is a basic technique to check HPLC peak homogeneity. It is also recommended that LC-MS be used while performing the peak purity.
Conclusion
I hope this article has helped you understand Peak Purity by PDA Detector and its importance. Now you can independently determine peak purity using a PDA detector during pharmaceutical method development.
You may also want to check out other articles on my blog, such as:
- Difference between HPLC and GC
- How to develop a method using GC-MS?
- Need of Chromatographic Method in Drug Development
- How to develop the HPLC method for acidic compounds?
- How to develop the HPLC method for non-polar compounds?
- What should be the Analytical method development approach?
- LC-MS in pharmaceutical development
- Tips on HPLC column equilibration and
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FAQs
What is a PDA or DAD detector?
The PDA detector acquires data simultaneously in UV and visible light. There the peak can be extracted at any wavelength of the wavelength range selected using the PDA detector.
What is the difference between UV and PDA detectors?
The main difference between UV and PDA detectors is that UV detector work at single wavelength where as the PDA detector work at the multiple wavelengths
What is the range of PDA in HPLC?
The range of PDA is UV and visible light (190 nm to 900 nm).
What is PDA scan?
PDA scan is 190 nm to 900 nm
Why is 254 nm used in HPLC?
Initially, the light source of the UV detector was a mercury lamp. This lamp was used in detectors with a fixed wavelength of 254 nm because it has a bright line at 253.7 nm (a wavelength with extremely high energy).
What is a dad detector?
DAD detector acquires data in the UV and visible ranges (190 nm to 900 nm) simultaneously .
How to determine peak purity?
For peak purity, spectra of at least three points upslope, peak maxima and downslope of any peak are taken and compared.A peak fails or passes the purity test depending on the match to the peak’s spectrum. Spectra of more than three points can also be collected and compare
What is the use of PDA detector in HPLC?
In HPLC, a PDA detector is used to extract the chromatogram at multiple wavelengths in the single run
What detector is used in HPLC for peak purity?
PDA detector and mass detectors are used in HPLC for peak purity
What is the range of the dad detector?
190 nm to 900 nm
What is peak purity concept?
According to the purity concept a peak fails or passes the purity test depending on the matching of the spectrum of the peak on the upslope, peak maxima and downslope.
What are the different methods of calculating peak purity?
Three methods; Spectral normalisation method, spectral similarity method and ratio gram generation method are widely used in calculating peak purity
Why is peak purity important in HPLC?
It tells about the co-elution of two or more peaks
Can peak purity of an inorganic compounds be performed by PDA?
No
What will be the peak purity if two chiral isomers are emitted at the same retention time?
The Peak purity result will be pure since most of the chiral isomers have the same UV spectrum
What is the difference between PDA and DAD Detector?
Both are the same.
What is the peak purity?
The process of assessing the spectral uniformity of a chromatographic peak is called the peak purity.
References
Abbreviations
- UV: Ultra-violet
- PDA: Photodiode array
- DAD: Diode array detector
- QL: Quantitation limit